Project Summary Recent research in evolutionary biology has begun to untangle the historical paths and mechanisms underlying how new molecular functions evolve. However, considerably less is known about why a particular path is taken by evolution or why a specific biochemical mechanism is used to evolve the new function in the first place. Was this the only path and mechanism available to evolution? Or did alternative paths and mechanisms exist, but were simply more difficult for evolution to find? Is selection capable of driving evolution towards a particular outcome or along a particular path, or is the result of evolution fundamentally dependent on chance events? What are the forces that determine the number of alternative paths and mechanisms available to evolution? Could other functions have evolved instead? To answer these questions, we need to know not only what has occurred during evolution, but also what else was possible. This project will address these questions using steroid receptor proteins as a model system. These proteins play important roles in human physiology and development and underlie risk of several human cancer types. As a consequence, the paths and mechanisms towards altered molecular function of steroid receptor proteins are of general medical relevance. To identify alternative outcomes and paths towards new molecular functions, a recently developed system for locus specific experimental evolution will be used to evolve an ancestral steroid receptor protein towards its modern function within the laboratory. By using an ancestral protein, this project will ?replay the tape of life? and explore multiple alternative evolutionary outcomes of an important transition in DNA binding specificity that underlies modern steroid receptors ability to activate distinct sets of genes in response to distinct steroid cues. In addition to determining how else the historical change in DNA specificity could have arisen, this project will evolve the same ancestral protein towards changes in DNA specificity that did not, but could have, occurred in nature to identify alternative paths that evolution may have taken given another opportunity. Using these evolved paths, the biochemical mechanisms underlying the changes in specificity will be determined and compared to the mechanism historically used by evolution. Finally, the sites of substitution, the types of substitution, and the mechanisms underlying change in DNA specificity will be compared among and between alternative paths and alternative changes in specificity to identify the determinants of why evolution found the particular outcome, took the particular path, and used the particular mechanism that it did. Answers to these questions will create a deeper understanding of how the evolutionary process works and help determine the extent to which changes in molecular function are restricted by the shape of sequence space and the evolutionary process.